This invention relates to a specific modified oligonucleotide complementary to a section of the human Ha-ras gene and mRNA, and its use to specifically regulate, modulate or inhibit expression of the Ha-ras gene, and its use as a pharmaceutical for the treatment of conditions arising from abnormal expression of the Ha-ras gene.
Antisense oligonucleotides (AO) have proven to be specific inhibitors of gene expression in a large number of systems, both in vivo and in vitro. (Uhlmann and Peyman, Chem. Rev. 1990, 90, 543).
One of the major problems encountered when using unmodified oligonucleotides containing only phosphodiester internucleoside linkages (PO-oligonucleotides) is the rapid degradation of this type of oligonucleotide in cells and biological fluids, such as, for example, serum and cerebrospinal fluid, by a range of nucleolytic activities. A wide range of chemical modifications to oligonucleotides have been carried out in order to improve their nucleolytic stability (Uhlmann and Peyman, Chem. Res. 1990, 90, 543). These modifications include the modification or replacement of the phosphodiester internucleoside linkage, the sugar unit, the nucleobase; or the sugar-phosphate backbone of the oligonucleotides. The most thoroughly investigated type of modification is alteration of the internucleoside linkage, including phosphorothioate (PS), methylphosphonate (MeP) and phosphorodithioate (PSS) linkages. It should be stressed that modification of the oligonucleotide alters not only its nuclease stability but also other characteristics of the oligonucleotide, such as, for example, their cellular uptake, RNaseH activation, and the strength and specificity of binding to their target nucleic acid, and the like. It should be borne in mind that the stability of the modified oligonucleotide in serum, frequently used to determine the nuclease stability of the oligonucleotide, is not the sole determinant of intracellular activity (P. D. Cook in xe2x80x9cAntisense Research and Applicationsxe2x80x9d, Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993, Chap.9, p149 et seq.).
The phosphorothioate (PS) modified oligonucleotides are the most widely used type of modified oligonucleotide. The following strategies have been developed for the positioning of PS linkages in antisense oligonucleotides:
(1) Replacement of all phosphodiester internucleoside linkages with phosphorothioate linkages.
The resulting all-phosphorothioate oligonucleotides are much more stable to nucleases than PO-oligonucleotides (Monia et al. J. Biol. Chem. 1996, 271, 14533). For example, degradation of all-PS oligonucleotides by endonucleases is slowed down by a factor of 2-45 relative to a PO oligonucleotide (Stein et al. Nucleic Acids Res. 1988, 16, 3209). In Xenopus oocytes or embryos, the degradation of microinjected PO oligonucleotides proceeds with a half-life of 30 minutes, while all-PS oligonucleotides have a half-life of over 3 hours under the same conditions (Woolf et al. Nucleic Acids Res. 1990, 18, 1763). All-PS oligonucleotides retain their ability to activate RNaseH. The major disadvantages of all-PS oligonucleotides are that their ability to form stable hybrids with their target nucleic acid is reduced, and that they frequently give rise to unspecific xe2x80x9cnon-antisensexe2x80x9d effects (Monia et al., J. Biol. Chem. 1996, 271, 14533).
(2) Oligonucleotides containing both phosphorothioate and phosphodiester internucleoside linkages.
In an effort to overcome the non-antisense effects observed with all-PS oligonucleotides, oligonucleotides containing both phosphorothioate and phosphodiester internucleoside linkages have been synthesized and tested for stability and biological activity.
Ghosh et al. (Anticancer Drug Design 1993, 8, 15) describe a PSxe2x80x94PO oligonucleotide containing various percentages of PS linkages. Their construction follows, for example, the pattern (PSxe2x80x94POxe2x80x94POxe2x80x94PO)n, (POxe2x80x94POxe2x80x94PS)n, (PSxe2x80x94PO)n, [(PO)2xe2x80x94(PS)2]n, [POxe2x80x94PSxe2x80x94PS]n. They teach that a PS linkage content of at least 50% is required for selective translation inhibition in vitro and that activity drops drastically when the PS content is less than 50%. More recently it has been demonstrated that an oligonucleotide containing 50% PS-linkages arranged in the pattern (PSxe2x80x94PO)n showed no biological activity in an assay system where an all-PS oligonucleotide and xe2x80x9cend-cappedxe2x80x9d POxe2x80x94PS oligonucleotides (see below) of the same sequence were highly active (Monia et al., J. Biol. Chem. 1996, 271, 14533).
(3) xe2x80x9cEnd-cappedxe2x80x9d Oligonucleotides, where one, two or three internucleoside bridges on the 5xe2x80x2 and/or the 3xe2x80x2 end of the oligonucleotide are phosphorothioate modified. (also known as the xe2x80x9cgap techniquexe2x80x9d)
This type of modification is designed primarily to protect the oligonucleotide from degradation by exonucleases. In particular modifications at the 3xe2x80x2-end of the oligonucleotide are desirable as they offer protection from 3xe2x80x2-exonucleases, which are the most abundant nucleases in serum (Uhlmann and Peyman, Chem. Rev. 1990, 90, 543).
An interesting comparison of strategies is found in Hoke et al. (Nucleic Acids Res. 1991, 19, 5743). The authors compare the activity of a range of antisense PS-oligonucleotides against HSV-1 in cell culture. Their findings confirm that 3xe2x80x2, or 3xe2x80x2+5xe2x80x2, end-capped oligonucleotides (the first three internucleoside linkages being modified in each case), similarly to all-PS oligonucleotides are sufficiently protected against degradation by nucleases in serum. In contrast internally modified (three PS bridges) oligonucleotides and oligonucleotides in which only the 5xe2x80x2-end has been capped (again, the first three internucleoside linkages being modified) are degraded rapidly. In contrast, the authors found that neither 5xe2x80x2 nor 3xe2x80x2 end capping nor both are sufficient for activity within the cell, and they drew the conclusion that a uniform modification (all-PS) is required to achieve sufficient stability to nucleases in cells.
More recently it has been discovered that pyrimidine nucleosides are the most nuclease susceptible points in oligonucleotides (Peyman, A. and Uhlmann, E., Biol. Chem. Hoppe-Seyler 1996, 377, 67; EP 0 653 439 A2). It was found that a combination of end-capping and PS protection of the pyrimidine positions of oligonucleotides (the so called xe2x80x9cminimal modificationxe2x80x9d approach) is sufficient to make them highly resistant to nuclease degradation. The biological activity (against Herpes simplex virus) of an oligonucleotide with this type of PS modification pattern was comparible to that of an all-PS oligonucleotide.
One of the major problems encountered when using AOs, whether or not they are stabilized against degradation, is their poor cellular uptake. Many approaches have been tried to attempt to ameliorate this problem. Most of these approaches involve the attachment of a variety of substances to the oligonucleotide. Modifications include: the attachment of peptides to oligonucleotides (Lemaitre, M. et al. Proc. Natl. Acad. Sci. USA 1987, 84, 648) and the attachment of lipophilic residues, such as alkyl chains or cholesterol, to oligonucleotides (Saison-Behmoaras, T. et al. EMBO J. 1991, 10, 1111-1118; Will, D. W. and Brown, T. Tetrahedron Lett. 1992, 33, 2729). It has been found, however, that in many cases the introduction of a lipophilic group causes biological effects which are independent of the sequence of the oligonucleotide. Non-specific effects have been reported for cholesterol-oligonucleotide conjugates (Henderson, G. B. and Stein, C. A. Nucleic Acids Res. 1995, 23, 3726.), and for oligonucleotides attached to alkyl chains (Shea, R. G. et al. Nucleic Acids Res. 1990, 18, 3777). Saison-Behmoaras et al (EMBO J. 1991, 10, 1111-1118; WO 96/34008) have reported that a 9mer all-PO oligonucleotide derivatized with a 3xe2x80x2-dodecanol moiety and a 5xe2x80x2-acridine crosslinking agent, and antisense to mutated Ha-ras inhibited T24 human bladder carcinoma cell proliferation. No comparison of the antiproliferative activity of this oligonucleotide with that of the corresponding oligonucleotide without a dodecanol conjugate was made. The cellular uptake of the dodecanol oligonucleotide was reported to be 4 times that of the unmodified oligonucleotide in T24 cells. The acridine-dodecanol modified oligonucleotide had no effect on the proliferation of a human mammary cell line carrying an unmutated Ha-ras gene. This effect can either be attributed to the sequence specificity of the antisense oligonucleotide or to insufficient cellular uptake. Since the uptake of the acridine-dodecanol oligonucleotide was not determined in the human mammary cell line, the effect may be entirely, or at least partly due to the fact that the oligonucleotide was not taken-up by the human mammary cell line, and thus did not have an opportunity to exhibit non-sequence-specific effects on cell proliferation.
About 20% of human tumors have a mutation in one of the three ras genes (Ha-ras, Ki-ras, and N-ras) leading to over-expression of p21 protein which plays an important role in the transformed phenotype (Bos, T. L. 1988 Mutation Res. 1988, 195, 255). It has been reported that inhibition of different ras genes in different cell lines can either have no effect, or inhibit cell proliferation depending on which of the ras genes is controlling the cell proliferation (Chen et al. J. Biol. Chem., 1996, 271, 28259-65). It was suggested that differential modulation of individual ras genes may be an approach to inhibit tumor growth while minimising effects on normal cell growth. One approach to devising successful therapy of Ha-ras-induced tumors is to regulate, modulate or inhibit expression of the Ha-ras gene. Antisense oligodeoxynucleotides (oligonucleotides) have been shown to act as specific inhibitors of ras mRNA expression in cell-free systems, in transformed cells in culture (T. Saison-Behmoaras et al. EMBO J. 1991, 10, 1111-1118; Monia et al. J. Biol. Chem. 1996, 271, 14533), and in ras-activated tumors in vivo (Gray, G. D. et al. Cancer Research, 1973, 53, 577).
In order to modulate the expression of the ras-gene in wild type or in mutated form Brown et al. (Oncogene Res. 1989, 4, 243-252) propose the use of anti-ras oligodeoxy ribonucleoside methylphosphonates which are complementary to the initiation codon region of Balb-ras P21 mRNA (a mouse version of the human Ha-ras Gene). However, it is well known that methyl phosphonates show some major disadvantages compared to phosphorothioates, among other their poor cellular uptake.
Monia et al. (J. Biol. Chem. 267, 19954-19962, 1992; WO 92122651) disclose all-PS antisense oligonucleotides and antisense oligonucleotides containing various percentages of PS linkages (WO 94/08003) directed to the translation initiation site or to codon 12 of the human Ha-ras gene, which however, have the above-mentioned disadvantages concerning the cellular uptake and their poor stability.
Pirollo et al. (Biochem. Biophys. Research Comm. 230, 196-201, 1997) propose the use of anti-ras all-PS oligodeoxynucleotides in order to reverse radio resistance of cancer cells. However, these antisense oligonucleotides show the disadvantages (poor cellular uptake, unstability) as mentioned above.
Therefore, this invention aims to provide an oligonucleotide, modified to improve its stability and cell uptake, complementary to Ha-ras mRNA which specifically regulates, modulates or inhibits expression of the Ha-ras gene in the form of its wild type as well as in its mutated forms, and which can be used to inhibit the proliferation of cancer cells, to reverse radio-resistance in cancer cells, and to treat conditions arising from abnormal expression of the Ha-ras gene.
According to the invention, this problem is solved by providing a modified oligodeoxynucleotide of the sequence SEQ ID NO: 1 5xe2x80x2-TxAxTxTxCxCxGxTxCxAxT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R
wherein
x is o or s
A is 2xe2x80x2-deoxyadenosine,
G is 2xe2x80x2-deoxyguanosine,
C is 2xe2x80x2-deoxycytidine and
T is thymidine.
The modified oligodeoxynucleotide according to the invention is particularly
characterized in that SEQ ID NO:1 has one of the following nucleotide linkage
variations:
(a) 5xe2x80x2-TsAsToToCsCoGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(b) 5xe2x80x2-TsAsToToCsCoGsToCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(c) 5xe2x80x2-TsAoTsToCsCoGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(d) 5xe2x80x2-TsAoTsTsCsCoGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(e) 5xe2x80x2-TsAoTsToCsCsGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(f) 5xe2x80x2-TsAoTsTsCsCsGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(g) 5xe2x80x2-TsAsToToCsCsGsToCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(h) 5xe2x80x2-TsAoTsTsCsCsGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(i) 5xe2x80x2-TsAsTsTsCsCsGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(j) 5xe2x80x2-TsAsTsToCoCsGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(k) 5xe2x80x2-TsAsTsToCsCsGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(l) 5xe2x80x2-TsAsTsToCsCoGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(m) 5xe2x80x2-TsAsToTsCsCoGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(n) 5xe2x80x2-TsAsToToCsCsGoTsCoAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(o) 5xe2x80x2-TsAsToTsCsCoGoTsCoAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(p) 5xe2x80x2-TsAoTsTsCoCsGoTsCoAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(q) 5xe2x80x2-TsAsTsToCsCsGoToCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(r) 5xe2x80x2-TsAsTsToCoCsGoToCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
(s) 5xe2x80x2-ToAoTsToCsCoGoToCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R or
(t) 5xe2x80x2-TsAsTsTsCsCsGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R,
wherein all the variables o, s, R, m, n and q and A, G, C and T have the above-mentioned meanings.
The modified oligonucleotide according to the present invention is complementary to the DNA or RNA deriving from the human Ha-ras gene. The oligonucleotide is complementary to the translation initiation region of Ha-ras mRNA. Therefore it inhibits both wild-type and mutant ras expression resulting in inhibition of tumor cell proliferation. The oligonucleotide is modified to improve its stability and cell uptake characteristics. The oligonucleotide contains four to nine phosphorothioate linkages at certain positions which are especially vulnerable to attack by nucleases. The oligonucleotide is modified at the 3xe2x80x2-end with a C8-C21-alkyl or with a C1-C21-alkyl, preferably C16-alkyl chain, covalently attached through either a phosphodiester bridge, an oligoethyleneglycol phosphodiester linkage, or a glyceryl ether phosphodiester linkage.
A particularly preferred embodiment of the present invention is a modified oligodeoxynucleotide in which SEQ ID NO:1 has the linkage variation of 5xe2x80x2-TsAsToToCsCoGoTsCsAsT-3xe2x80x2-Oxe2x80x94PO2xe2x80x94Oxe2x80x94R (SEQ ID NO: 1), containing six phosphorothioate linkages, wherein the variables o, s, R, n, m, and q and A, G, C and T have the above-mentioned meanings.
R is particularly preferably xe2x80x94CH2CH(OH)CH2O(CH2)15CH3, xe2x80x94(CH2)15CH3 or xe2x80x94(CH2CH2O)nxe2x80x94(CH2)15CH3, wherein n is an integer from 1 to 6; preferably, n is 1, 2 or 3.
The invention further relates to the preparation of a modified oligonucleotide. Preferably the oligonucleotide is synthesized using standard phosphoramidite chamistry as outlind in examples 1 and 2. Phosphorothioate linkages are for example introduced by sulfurization using the Beaucage reagent.
Surprisingly it was found that particularly the glycerol alkyl ether, i.e. the group xe2x80x94CH2xe2x80x94CH(OH)CH2Oxe2x80x94(CH2)qxe2x80x94CH3, q being an integer from 7 to 20, in particular the group xe2x80x94CH2CH(OH)CH2O(CH2)15CH3, linked by a phosphodiester linkage to the 3xe2x80x2-end of the oligonucleotide renders an oligonucleotide which no longer depends on the mixing with uptake enhancers for optimal biological activity. This is the first time that an oligonucleotide has been found to exhibit the same biological activity with and without the addition of an uptake enhancer.
The invention further relates to the preparation of a modified oligonucleotide. Preferably the oligonucleotide is synthesized using standard phosphoramidite chemistry as outlined in examples 1 and 2. Phosphorsthioate linkages are for example introduced sulfuization using the Beaucage reagent.
The modified oligodeoxynucleotide according to the present invention as described above is particularly suitable for use as a pharmaceutical. Therefore, a further object of the present invention is the modified olidodeoxynucleotide as described above for use as a pharmaceutical, in particular a pharmaceutical formulation which contains an effective amount of at least one modified oligodeoxynucleotide according to the invention. More particularly, the oligodeoxynucleotide according to the present invention is suitable for preparing a pharmaceutical for the treatment of a disease arising from the overexpression and/or mutation of the Ha-ras gene or diseases arising from a hyperproliferative disorder. Such diseases are in particular cancer, restenosis, or psoriasis.
Transforming activation of ras occurs in approximately 10-20% of human tumors by amplification of the ras gene at the DNA level or by overexpression of the ras protein at the mRNA level. Ras genes can be activated by point mutation at codon 12, 13, or 61 (Barbacid, M. Ann. Rev. Biochem. 1987, 56, 779-827; Bos, supra; Lowy, supra). Point mutation of the Ha-ras gene at codon 12 converts the normally regulated protein to one that is continually active. It is thought that the loss of regulation of normal ras protein function is responsible for the transformation from normal to malignant cell growth.
The modified oligodeoxynucleotide according to the invention is particularly suitable for the regulation, modulation, or inhibition of Ha-ras gene expression. The oligodeoxynucleotide is complementary to the translation initiation region of Ha-ras mRNA. Therefore, it inhibits both wild-type and mutant ras expression resulting in inhibition of tumor cell proliferation. The oligodeoxynucleotide according to the invention is further modified to improve its stability and cell uptake characteristics. The oligodeoxynucleotide contains four to nine, preferably six, phosphorothioate linkages at certain positions which are especially vulnerable to attack by nucleases. The cellular uptake of the oligodeoxynucleotide is further improved by a C8-C21-alkyl group or by an C1-C21-alkyl group covalently attached through either a phosphodiester bridge, an oligoethyleneglycol phosphodiester linkage, or a glyceryl ether phosphodiester linkage. Due to the improved stability and to the improved cellular uptake the oligodeoxynucleotide according to the present invention depends no longer on the mixing with uptake enhancers and is rendered for optimal biological activity.
Therefore, the oligodeoxynucleotide according to the present invention is particularly suitable for preparing a pharmaceutical which is directed to the regulation modulation or inhibition of Ha-ras gene expression of both, of the Ha-ras wild type gene and its mutants.
In case that the disease resulting from mutation of the Ha-ras gene is cancer, the oligonucleotide according to the invention is particularly suitable for preparing a pharmaceutical which inhibits the proliferation of cancer cells.
However, on the other hand the oligodeoxy nucleotide is advantageously used for preparing a pharmaceutical against cancer which is used in combination with chemotherapy, in particular in cases where the cancer cells have become resistant to chemotherapy, since the oligodeoxynucleotide according to the invention reverses chemoresistance in cancer cells. Therefore, a further object of the present invention is the use of the modified oligodeoxynucleotide as described above for preparing a pharmaceutical which reverses the chemoresistance in cancer cells.
In particular due to this particular effect the oligonucleotide according to the present invention is suitable for preparing a pharmaceutical formulation which contains an effective amount of at least one further chemotherapeutically effective agent. Such chemotherapeutically effective agents are for example cis-platinum and its derivatives, preferably cis-platinum, N-lost derivatives, preferably cyclophosphamide, trofosfamide and ifosfamide, aziridine derivatives, preferably thiothepa, N-nitrosourea derivatives, folic acid antagonists, preferably methotrexate, analogues of purine and pyrimidine bases, preferably 5-fluoro-uracil, cytostatically effective antibiotics, preferably adriamycine, mitomycine and daunorubicine, estrogene antagonists, preferably tamoxifene, and nucleoside derivatives, preferably MDL 101, 731 ((E)-2xe2x80x2-deoxy-2xe2x80x2-(fluoromethylene)cytidine; Cancer Research 54, 1485-1490, 1994).
Ras genes are known to be involved in signal-transduction of various factors for growth, differentiation and oncogenesis. Recent studies have implicated the raf-1 oncogene, which is downstream from ras in the signal transduction pathway, in the expression of the radiation resistance phenotype. It can be shown that the antisense oligonucleotide according to the present invention which is directed to the initiation codon of Ha-ras reverses the radiation resistance level of cancer cells.
Therefore, a further object of the present invention is the use of the modified oligodeoxynucleotide according to the invention for preparing a pharmaceutical for the treatment of cancer in combination with radiotherapy. Particularly, the modified oligodeoxynucleotide according to the present invention is suitable for preparing a pharmaceutical which reverses the radioresistance in cancer cells.